Figure 2.

Fn matrix assembly regulates endothelial cell 3D morphogenesis. (A) Endothelial cells were suspended in 3D fibrin gels with FITC-labeled Fn in the absence or presence of the FUD peptide (250 nM), with matrix assembly and cell morphology monitored by confocal laser microscopy. Bar, 20 μm. (B) Endothelial cells were cultured within 3D fibrin matrices for 6 d in the presence or absence of Fn matrix inhibitors. Cell number was quantified following gel digestion with collagenase. (C) Endothelial cells were cultured in 3D fibrin gels under control conditions or in the presence of various Fn matrix assembly inhibitors for 6 d, and tubulogenesis was assessed by sectioning and H&E staining of the matrices followed by lumen quantification. (D) Endothelial cells spheroids were embedded in gels of 3D fibrin and cultured for 6 d in the absence or presence of mAb L8 (100 μg/uL), and tubulogenesis was assessed by phase contrast microscopy (left panels in the top and bottom series) or light microscopy (H&E-stained cross-sections in the middle column). (Right panels) The assembly of a FITC-labeled Fn matrix was monitored by confocal laser microscopy. Bar, 50 μm. (E) Endothelial cells were cultured within a matrix of type I collagen in the presence or absence of mAb L8 and tubulogenesis assessed at 6 d by phase contrast microscopy. Bar, 50 μm. (F) Endothelial cells were cultured atop 3D fibrin gels for 6 d in the presence of L8 or control IgG. (Inset) Assembly of FITC-Fn into a fibrillar matrix was monitored by fluorescence microscopy, and cell morphology was assessed by phase contrast microscopy. (G) Endothelial cell 2D migration was assessed atop a fibrin-coated substratum in the presence of IgG or L8 (arrowhead marks the edge of the monolayer at the start of the 2-d culture period). (H) Endothelial cell growth was monitored during a 6-d culture period in the presence of mAb L8, mAb 9D2, or the 70-kDa Fn fragment relative to an IgG control. Bar, 100 μm.